This proposal uses molecular genetic techniques to explore the basis of intracellular infection by Legionella pneumophila, the etiologic agent of Legionnaires' disease. After isolating mutations in genes that encode structures in the bacterial cell envelope of this organism, mutant will be analyzed for impaired infectivity in a tissue culture model of intracellular infection, and those found to be impaired will be tested for virulence in an animal model of infection. Mutations will be isolated by three complementary techniques: i) Site-specific mutations will be introduced into the genes for two previously cloned surface antigens of L. pneumophila using genetic techniques developed in this laboratory. ii) L. pneumophila mutagenized by random insertion of a mini-Mu transposon (Mud 4041) will be screened for loss of three phenotypes that may be associated with intracellular survival (loss of flagella or LPS O-side chains will be detected by in situ immunoassay using specific monoclonal antibodies and loss of acid phosphatase by an in situ enzyme assay). iii) Mutations in genes encoding secreted proteins of L. pneumophila will be constructed by shuttle mutagenesis with TnphoA (i.e., genes from a genomic cosmid library in E. coli that produce a PhoA+ fusion after insertion of TnphoA will be transferred to L. pneumophila and exchanged for the wild type gene). All mutants will be screened for loss of infectivity in the U937 cell model of intracellular infection. Genes found to be necessary for full infectivity will be used to reconstruct the mutation, by allelic exchange, in a virulent animal-passaged isolate of L. pneumophila. Then, this isolate will be compared in U937 cell model with each specific mutant and with each mutant after complementation (by reintroduction of the wild type gene) to confirm the attenuated phenotype. Mutants that are attenuated in tissue culture infection will then be compared with parent and complemented strains for lethality in intratracheally-inoculated guinea pigs. In addition to identifying factors necessary for the intracellular life cycle and animal virulence by answering "molecular Koch's postulates", these studies will provide significant additional evidence for the hypothesis that intracellular infection is a prerequisite for Legionnaires' disease.